The chitinolytic activity of Penicillium janthinellum P9: purification, partial characterization and potential applications

AIMS: To purify and characterize the chitinolytic activity of Penicillium janthinellum P9 and to evaluate possible uses of the purified enzymes in the control of fungal growth and spore germination. METHODS AND RESULTS: The chitinolytic activity of P. janthinellum P9 was associated to two beta-N-acetyl-hexosaminidases (CHI1 and CHI2) that were purified by preparative isoelectric focusing and preparative electrophoresis and partially characterized. Treatment of test fungi with purified enzyme solutions caused reduced spore germination, reduction of hyphal length and mycelial damage. The combined action of the two enzymes and a systemic fungicide completely inactivated pests and food-spoiling moulds such as Fusarium solanii, P. canescens and Cladosporium cladosporioides. Treatment with the two enzymes increased germination of freeze-dried fungal spores. CONCLUSION: The chitinolytic activity of P. janthinellum P9 is associated with two extracellular beta-N-acetyl-hexosaminidases that can cause damage to the cell walls of other fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This appears to be the first report on the characterization of extracellular chitinolytic enzymes produced by a Penicillium strain. The results of this study might have some impact in the applied research field.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Prevention by delay: nonspecific immunity elicited by IL-12 hinders Her-2/neu mammary carcinogenesis in transgenic mice

As a natural consequence of the expression of the activated transforming rat Her-2/neu oncogene all mammary glands of female transgenic BALB/c (BALB-neuT) mice develop atypical epithelial hyperplasia which progresses to invasive carcinoma. A lobular carcinoma is palpable in all mammary glands of 33-week-old BALB-neuT mice. This progression is markedly delayed by systemic administration of IL-12. In a series of studies the best administration schedule, the lowest dose and the most effective administration time have been defined. The cellular and molecular mechanisms resulting in the delay of carcinogenesis have been established. By means of a series of downstream mediators IL-12 inhibits the angiogenic burst that goes along with the passage from preneoplastic to neoplastic and invasive lesions; it also recruits lymphoid cells in the mammary pad and activates their cytotoxicity towards neoplastic cells and newly formed vessels; and furthermore, it induces lymphoid cells to trigger antiangiogenic activities in neoplastic epithelial cells. Effective, low-dose and non-toxic IL-12 treatments may thus be envisaged as a possible option in the management of preneoplastic mammary lesions and in mammary cancer prevention.     

Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding

Na+ binding to thrombin enhances the catalytic activity toward numerous synthetic and natural substrates. The bound Na+ is located in a solvent channel 16 A away from the catalytic triad, and connects with D189 in the S1 site through an intervening water molecule. Molecular modeling indicates that the G184K substitution in thrombin positions the protonated epsilon-amino group of the Lys side-chain to replace the bound Na+. Likewise, the G184R substitution positions the guanidinium group of the longer Arg side-chain to replace both the bound Na+ and the connecting water molecule to D189. We explored whether the G184K or G184R substitution would replace the bound Na+ and yield a thrombin derivative stabilized in the highly active fast form. Both the G184K and G184R mutants lost sensitivity to monovalent cations, as expected, but their activity toward a chromogenic substrate was compromised up to 200-fold as a result of impaired diffusion into the S1 site and decreased deacylation rate. Interestingly, both G184K and G184R substitutions compromised cleavage of procoagulant substrates fibrinogen and PAR1 more than that of the anticoagulant substrate protein C. These findings demonstrate that Na+ binding to thrombin is difficult to mimic functionally with residue side-chains, in analogy with results from other systems.     

Role of superoxide anion in regulating pressor and vascular hypertrophic response to angiotensin II

Our purpose was to address the role of NAPDH oxidase-derived superoxide anion in the vascular response to ANG II. Blood pressure, aortic superoxide anion, 3-nitrotyrosine, and medial cross-sectional area were compared in wild-type mice and in mice that overexpress human superoxide dismutase (hSOD). The pressor response to ANG II was significantly less in hSOD mice. Superoxide anion levels were increased twofold in ANG II-treated wild-type mice but not in hSOD mice. 3-Nitrotyrosine increased in aortic endothelium and adventitia in wild-type but not hSOD mice. In contrast, aortic medial cross-sectional area increased 50% with ANG II in hSOD mice, comparable to wild-type mice. The lower pressor response to ANG II in the mice expressing hSOD is consistent with a pressor role of superoxide anion in wild-type mice, most likely because it reacts with nitric oxide. Despite preventing the increase in superoxide anion and 3-nitrotyrosine, the aortic hypertrophic response to ANG II in vivo was unaffected by hSOD.     

Protein aggregation in Escherichia coli: role of proteases

Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases.     

A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein

A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.